Method for chemoprevention of prostate cancer

ABSTRACT

This invention provides the chemoprevention of prostate cancer and, more particularly, to a method of preventing prostate carcinogenesis comprising the steps of administering to a human subject having a precancerous precursor of prostate adenocarcinoma, a pharmaceutical preparation comprising a chemopreventive agent to prevent, prevent recurrence of, suppress or inhibit prostate carcinogenesis. The present invention provides a safe and effective method for suppressing or inhibiting latent prostate cancer and is particularly useful for treating subjects having elevated risk of developing prostate cancer, for example, those having benign prostatic hyperplasia, prostate intraepithelial neoplasia (PIN), or an abnormally high level of circulating prostate specific antibody (PSA), or who have a family history of prostate cancer.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a Continuation-in-Part Application of U.S. Ser. No.09/436,208, filed Nov. 8, 1999, which is a Continuation-in-PartApplication of U.S. Ser. No. 09/306,958, filed May 7, 1999, now U.S.Pat. No. 6,265,448 which claims priority of U.S. Provisional ApplicationNo. 60/084,602, filed May 7, 1998, which is hereby incorporated byreference in its entirety.

FIELD OF INVENTION

This invention relates to the chemoprevention of prostate cancer and,more particularly, to a method of administering to a subject aneffective dose of an antiestrogen agent to prevent and prevent therecurrence of, suppression or inhibition of prostate carcinogenesis.

BACKGROUND OF THE INVENTION

Prostate cancer is one of the most frequently occurring cancers amongmen in the United States, with hundreds of thousands of new casesdiagnosed each year. Unfortunately, over sixty percent of newlydiagnosed cases of prostate cancer are found to be pathologicallyadvanced, with no cure and a dismal prognosis. One approach to thisproblem is to find prostate cancer earlier through screening programsand thereby reduce the number of advanced prostate cancer patients.Another strategy, however, is to develop drugs to prevent prostatecancer. One third of all men over 50 years of age have a latent form ofprostate cancer that may be activated into the life-threatening clinicalprostate cancer form. The frequency of latent prostatic tumors has beenshown to increase substantially with each decade of life from the 50s(5.3-14%) to the 90s (40-80%). The number of people with latent prostatecancer is the same across all cultures, ethnic groups, and races, yetthe frequency of clinically aggressive cancer is markedly different.This suggests that environmental factors may play a role in activatinglatent prostate cancer. Thus, the development of chemopreventionstrategies against prostate cancer may have the greatest overall impactboth medically and economically against prostate cancer.

Because of the high incidence and mortality of prostate cancer, it isimperative to develop chemoprevention strategies against thisdevastating disease. Understanding those factors that contribute toprostate carcinogenesis including the initiation, promotion, andprogression of prostate cancer will provide molecular mechanistic cluesas to appropriate points of intervention to prevent or halt thecarcinogenic process. New innovative approaches are urgently needed atboth the basic science and clinical levels to decrease the incidence ofprostate cancer as well as to halt or cause the regression of latentprostate cancer. As the frequency of prostate cancer escalatesdramatically at the same ages when men are confronted by other competingcauses of mortality, simply slowing the progression of prostateadenocarcinoma may be both a more suitable and cost effective healthstrategy.

Various approaches have been taken to the chemoprevention of prostatecancer. Greenwald, “Expanding Horizons in Breast and Prostate CancerPrevention and Early Detection” in J. Cancer Education, 1993, Vol. 8,No. 2, pages 91-107, discusses the testing of 5α-reductase Inhibitorssuch as finasteride for the prevention of prostate cancer. Brawley etal., “Chemoprevention of Prostate Cancer” in Urology, 1994, Vol. 43, No.5, also mentions 5α-reductase inhibitors as well asdifluoromethylornithine and retinoids as potential chemopreventiveagents.

Kelloff et al., “Introductory Remarks: Development of ChemopreventiveAgents for Prostate Cancer” in Journal of Cellular Biochemistry, 1992,Supplement 16H: 1-8, describes National Cancer Institute preclinicalstudies of seven agents: all-trans-N-(4-hydroxyphenyl)retinamide,difluoromethylornithine, dehydroepiandrosterone, liarozole, lovestatin,oltipraz, and finasteride.

Lucia et al., “Chemopreventive Activity of Tamoxifen,N-(4-Hydroxyphenyl)retinamide, and the Vitamin D Analogue Ro24-553 1 forAndrogen-promoted Carcinomas of the Rat Seminal Vesicle and Prostate” inCancer Research, 1995, Vol. 55, pages 5621-5627, reports chemopreventionof prostate carcinomas in Lobund-Wistar rats by tamoxifen, an estrogenresponse modifier.

As discussed in Potter et al., “A mechanistic hypothesis for DNA adductformation by tamoxifen following hepatic oxidative metabolism” inCarcinogenesis, 1994, Vol. 15, No. 3, pages 439-442, tamoxifen causesliver carcinogenicity in rats, which is attributed to the formation ofcovalent DNA adducts. This reference also reports that the tamoxifenanalogue toremifene, which showed a much lower level of hepatic DNAadduct formation than tamoxifen, is non-carcinogenic.

Toremifene is an example of a triphenylalkene compound described In U.S.Pat. Nos. 4,696,949 and 5,491,173 to Toivola et al., the disclosures ofwhich are incorporated herein by reference. The parenteral and topicaladministration to mammalian subjects of formulations containingtoremifene are described in U.S. Pat. No. 5,571,534 to Jalonen et al.and in U.S. Pat. No. 5,605,700 to DeGregorio et al., the disclosures ofwhich are incorporated herein by reference.

Torernifene-containing formulations for reversing the multidrugresistance to cancer cells to a cytotoxic drug are described in U.S.Pat. No. 4,990,538 to Harris et al., the disclosure of which isincorporated herein by reference. U.S. Pat. Nos. 5,595,722 and 5,599,844to Grainger et al., the disclosures of which are incorporated herein byreference. describe methods for Identifying agents that increase TGFPlevels and for orally administering formulations containing TGFPactivators and TGFP production stimulators to prevent or treatconditions characterized by abnormal proliferation of smooth musclecells, for example, vascular trauma. Disclosed agents for increasingTGFP levels Include tamoxifen and its analogue toremifene.

U.S. Pat. Nos. 5,629,007 and 5,635,197 to Audia et al., the disclosuresof which are incorporated herein by reference, describe a method ofpreventing the development of prostatic cancer at risk of developingsuch cancer, for example, a patient having benign prostatic hyperplasia,by administering to the patient an octahydrobenzo[f}quinolin-3-onecompound.

U.S. Pat. No. 5,595,985 to Labrie, the disclosure of which isincorporated herein by reference, also describe a method for treatingbenign prostatic hyperplasia using a combination of a 5α-reductaseinhibitor and a compound that binds and blocks access to androgenreceptors. One example of a compound that blocks androgen receptors isflutamide.

U.S. Pat. Nos. 4,329,364 and 4,474,813 to Neri et al., the disclosuresof which are incorporated herein by reference, describe pharmaceuticalpreparations comprising flutamide for delaying and/or preventing theonset of prostate carcinoma. The preparation can be in the form of acapsule, tablet, suppository, or elixir. Despite these developments,there is a continuing need for agents and methods effective forpreventing prostate cancer. The present invention is directed tosatisfying this need.

SUMMARY OF THE INVENTION

This invention provides the chemoprevention of prostate cancer and, moreparticularly, to a method of administering to a subject an effectivedose of an antiestrogen to prevent, prevent recurrence of, and/orsuppress or inhibit prostate carcinogenesis.

This invention provides the chemoprevention of prostate cancer and, moreparticularly, to a method of administering to a subject an effectivedose of an antiestrogen which does not cause the formation of DNAadducts, to prevent, prevent recurrence of, and/or suppress or inhibitprostate carcinogenesis.

This invention provides the chemoprevention of prostate cancer and, moreparticularly, to a method of administering to a subject an effectivedose of a chemopreventive agent, toremifene and analogs or metabolitesthereof, to prevent, prevent recurrence of, and/or suppress or inhibitprostate carcinogenesis.

The present invention is directed to a method for preventing prostatecarcinogenesis with an antiestrogen which does not cause the formationof DNA adducts. Further, this invention involes administering to amammalian subject a pharmaceutical preparation of a chemopreventiveagent having the formula:

wherein R₁ and R₂, which can be the same or different, are H or OH, R₃is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different,are H or an alkyl group of 1 to about 4 carbon atoms; and theirpharmaceutically acceptable carrier, diluents, salts, esters, orN-oxides, and mixtures thereof.

The present invention provides a safe and effective method forsuppressing or inhibiting latent prostate cancer and is particularlyuseful for treating subjects having an elevated risk of developingprostate cancer, for example, those having benign prostatic hyperplasia,prostate intraepithelial neoplasia (PIN), or an abnormally high level ofcirculating prostate specific antibody (PSA), or who have a familyhistory of prostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A graph illustrating the chemopreventive effects of toremifenein the TRAMP model.

FIGS. 2A-2C: H&E sections illustrating ventral prostate cells in normalmice and prostate carcinoma in TRAMP mice included in the study.

FIG. 3: Effect of Toremifene on ventral prostate development in theTRAMP mouse.

FIG. 4: Effect of Toremifene on tumor occurrence in the TRAMP mice.

FIG. 5: Effect of Toremifene on tumor development in the TRAMP model.

FIG. 6: Comparison of placebo vs. Toremifene effects on tumor growth.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides the chemoprevention of prostate cancer and, moreparticularly, to a method of administering to a subject an effectivedose of an antiestrogen which does not cause the formation of DNAadducts to prevent, prevent recurrence of, and/or suppress or inhibitprostate carcinogenesis.

This invention provides a method for preventing prostate carcinogenesis;2) methods for suppressing or inhibiting prostate cancer; 3) methods forreducing the risk of developing prostate cancer; and 4) methods forincreasing the survival rate of a subject using an antiestrogen whichdoes not cause the formation of DNA adducts as the prostatechemopreventive.

As demonstrated herein, toremifene is one example of an antiestrogenwhich does not cause the formation of DNA adducts which is a prostatechemopreventive agent. In the experiments conducted herein in bothanimal and human studies the antiestrogen, was shown to prevent prostatecancer. The prostates were actually dissected and evaluated bothhistologically and by wholemount analysis. Also, Toremifene was testedfor the prevention of prostate cancer by treating LNCaP xenografts innude mice. As is shown, the data is quite dramatic, not only has anantiestrogen such as toremifene inhibited growth, but actuallytoremifene was able to produce regression of the tumors. Further, inhuman studies conducted with the antiestrogen, high grade PIN (HGPIN)which has been established and time tested as a precursor lesion forhuman prostate cancer also known as latent prostate cancer, has shownregression. Thus, demonstrating that the antiestrogen toremifene is aprostate chemopreventive agent.

The present invention is directed to a method for preventing prostatecarcinogenesis. Antiestrogens which act as prostate chemopreventiveagents include but are not limited to: toremifene and analogs orsynthetics thereof; selective estogen receptor modulators (SERMS),triphenylethylenes which include droloxifene, idoxifene,(2)-4-OH-tamoxifene, chromans such as levomeloxifene, and centchroman;benzothiophenes such as raloxifene, and LY 353381; naphthalens such asCP336,156; phytoestrogens such as isoflavanoids including daidzein,genistein, yenoestrogens; coumestrol; zearalenone; daidzein; apigenin;waempferol; phloretin; biochanin A; naringenin; formononetin;ipriflavone; quercetin; chrysin; flavonoids; flavones, isoflavones,flavanones, and chalcones); coumestans; mycoestrogens; resorcyclic acidlactone; nafoxideneand equol, and lignan including enterodiol andenterolactone; and other compounds which are known in the art asfollows: ICI 164,384, ICI 182, 780; TAT-59, EM-652 (SCG 57068), EM-800(SCH57050), EM-139, EM-651, EM-776, and peptide antagonist of humanestrogen receptors. This invention involves administering to a subject apharmaceutical preparation of a chemopreventive agent having theformula:

wherein R₁ and R₂, which can be the same or different, are H or OH, R₃is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different,are H or an alkyl group of 1 to about 4 carbon atoms; and theirpharmaceutically acceptable carrier, diluents, salts, esters, orN-oxides, and mixtures thereof.

This invention provides the use of a pharmaceutical composition forpreventing prostate cancer, the recurrence of, suppression or inhibitionof prostate carcinogenesis, or increasing the survival rate of a subjecthaving prostate cancer, comprising an antiestrogen which does not causethe formation of DNA adducts and a suitable diluent. The antiestrogensinclude the antiestrogens provided above.

This invention provides for the use of a pharmaceutical composition forpreventing prostate cancer, the recurrence of, suppression or inhibitionof prostate carcinogenesis, or increasing the survival rate of a subjecthaving prostate cancer, comprising a chemopreventive agent having theformula:

wherein R₁ and R₂, which can be the same or different, are H or OH, R₃is OCH₂CH₂NR₄R₅, wherein R4 and R₅, which can be the same or different,are H or an alkyl group of 1 to about 4 carbon atoms; and theirpharmaceutically acceptable carrier, diluents, salts, esters, orN-oxides, and mixtures thereof.

The present invention provides a safe and effective method forsuppressing or inhibiting latent prostate cancer and is particularlyuseful treating subjects having an elevated risk of developing prostatecancer, for example, those having benign prostatic hyperplasia, prostateintraepithelial neoplasia (PIN), or an abnormally high level ofcirculating prostate specific antibody (PSA), or who have a familyhistory of prostate cancer. Prostate cancer shall include preventingprostate carcinogenesis; suppressing or inhibiting prostate cancer;reducing the risk of developing prostate cancer; increasing the survivalrate of a subject with prostate cancer; and treating prostate cancer.

The compound 4-chloro-1,2-diphenyl-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl[-1-butene of formula (I), where R₁ and R₂ are each H andR₄ and R₅ are each methyl, is named toremifene. Toremifene has beenshown safe and effective as an anti-tumor compound. On administration,toremifene has several metabolites that are also biologically active.

This invention also provides for use of toremifene analogs ormetabolites thereof, which are well known to those skilled in the art.Other examples of chemopreventive agents of formula (I) are thefollowing:4-chloro-1,2-diphenyl-1-[4-[2-(N-methylamino)ethoxy]phenyl]-1-butene;4-chloro-1,2-diphenyl-1-[4-[2-(N,N-diethylamino)ethoxy]phenyl]-1-butene;4-chloro-1,2-diphenyl-1-[4-(aminoethoxy)phenyl]-1-butene;4-chloro-1,2-diphenyl-1-[4-(aminoeethoxy)phenyl]-1-butene;4-chloro-1-(4-hydroxyphenyl)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-2-phenyl-1-butene;4-chloro-1-(4-hydroxyphenyl)-1-[4-[2-(N-methylamino)ethoxy]phenyl]-2-phenyl-1-butene;and4-chloro-1,2-bis(4-hydroxyphenyl)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-butene.

The invention encompasses pure (Z)- and (E)-isomers of the compounds andmixtures thereof as well as pure (RR,SS)- and (RS,SR)-enantiomer couplesand mixtures thereof.

The agent compounds of formula (I) can be prepared according toprocedures described in the previously cited U.S. Pat. Nos. 4,696,949and 5,491,173 to Toivola et al.

The invention includes pharmaceutically acceptable salts ofamino-substituted compounds with organic and inorganic acids, forexample, citric acid and hydrochloric acid. The invention also includesN-oxides of the amino substituents of the compounds of formula (I).Pharmaceutically acceptable salts can also be prepared from the phenoliccompounds by treatment with inorganic bases, for example, sodiumhydroxide. Also, esters of the phenolic compounds can be made withaliphatic and aromatic carboxylic acids, for example, acetic acid andbenzoic acid esters.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the agent together with suitable diluents,preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A“therapeutically effective amount” as used herein refers to that amountwhich provides a therapeutic effect for a given condition andadministration regimen. Such compositions are liquids or lyophilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts).solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines). Other embodiments of thecompositions of the invention incorporate particulate forms protectivecoatings, protease inhibitors or permeation enhancers for various routesof administration, including parenteral, pulmonary, nasal and oral. Inone embodiment the pharmaceutical composition is administeredparenterally, paracancerally, transmucosally, transdermally,intramuscularly, Intravenously, intradermally, subcutaneously,intraperitonealy, intraventricularly, intracranially and intratumorally.The dosage may be in the range of 20-80 mg/day. In another embodimentthe dosage is in the range of 35-66 mg/day. In another embodiment thedosage is in the range of 40-60 mg/day. In another embodiment the dosageis in a range of 45-60 mg/day. The dosage may be 40-45 mg/day. Thedosage may be 60 mg/day. The dosage may be 45 mg/day.

Further, as used herein “pharmaceutically acceptable carrier” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphold system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvant include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvant such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral. Compounds modified by the covalent attachment of water-solublepolymers such as polyethylene glycol, copolymers of polyethylene glycoland polypropylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinylpyrrolidone or polyproline are known to exhibitsubstantially longer half-lives in blood following intravenous injectionthan do the corresponding unmodified compounds (Abuchowski et al., 1981;Newmark et al., 1982; and Katre et al., 1987). Such modifications mayalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. As a result, the desired in vivo biological activity may beachieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. For example, the agent may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment,polymeric materials can be used. In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984). Preferably, a controlled releasedevice is introduced into a subject in proximity of the site ofinappropriate immune activation or a tumor. Other controlled releasesystems are discussed in the review by Langer (Science 249:1527-1533(1990).

The method of the present invention for preventing prostatecarcinogenesis involves administering to a mammalian subject apharmaceutical preparation comprising chemopreventive agent or ametabolite or salt thereof. The pharmaceutical preparation can comprisethe chemopreventive agent alone, or can further include apharmaceutically acceptable carrier, and can be in solid or liquid formsuch as tablets, powders, capsules, pellets, solutions, suspensions,elixirs, emulsions, gels, creams, or suppositories, including rectal andurethral suppositories. Pharmaceutically acceptable carriers includegums, starches, sugars, cellulosic materials, and mixtures thereof. Thepharmaceutical preparation containing the chemopreventive agent can beadministered to a subject by, for example, subcutaneous implantation ofa pellet; in a further embodiment, the pellet provides for controlledrelease of chemopreventive agent over a period of time. The preparationcan also be administered by intravenous, intraarterial, or intramuscularinjection of a liquid preparation, oral administration of a liquid orsolid preparation, or by topical application. Administration can also beaccomplished by use of a rectal suppository or a urethral suppository,The pharmaceutical preparation can also be a parenteral formulation; inone embodiment, the formulation comprises a liposome that includes acomplex of a chemopreventive agent such as, for example, toremifene anda cyclodextrir compound, as described in the previously cited U.S. Pat.No. 5,571,534 to Jalonen et al.

The pharmaceutical preparations of the invention can be prepared byknown dissolving, mixing, granulating, or tablet-forming processes. Fororal administration, the chemopreventive agents or their physiologicallytolerated derivatives such as salts, esters, N-oxides, and the like aremixed with additives customary for this purpose, such as vehicles,stabilizers, or inert diluents, and converted by customary methods intoa suitable form for administration, such as tablets, coated tablets,hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.Examples of suitable inert vehicles are conventional tablet bases suchas lactose, sucrose, or cornstarch in combination with binders likeacacia, cornstarch, gelatin, or with disintegrating agents such ascornstarch, potato starch, alginic acid, or with a lubricant likestearic acid or magnesium stearate. Examples of suitable oily vehiclesor solvents are vegetable or animal oils such as sunflower oil orfish-liver oil. Preparations can be effected both as dry and as wetgranules. For parenteral administration (subcutaneous, intravenous,intraarterial, or intramuscular injection), the chemopreventive agentsor their physiologically tolerated derivatives such as salts, esters,N-oxides, and the like are converted into a solution, suspension, oremulsion, if desired with the substances customary and suitable for thispurpose, for example, solubilizers or other auxiliaries. Examples are:sterile liquids such as water and oils, with or without the addition ofa surfactant and other pharmaceutically acceptable adjuvants.Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solutions,and glycols such as propylene glycols or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with exciplents which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

For topical administration to body surfaces using, for example, creams,gels, drops, and the like, the chemopreventive agents or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds), Liss, N.Y., pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

The pharmaceutical compositions of the present invention areparticularly useful for treating a subject having an elevated risk ofdeveloping prostate cancer. High-risk subjects include, for example,those having benign prostatic hyperplasia, prostatic intraepithelialneoplasia (PIN), or an abnormally high level of circulating prostatespecific antibody (PSA), or who have a family history of prostatecancer.

Further, the prostate chemopreventive agent may be administered incombination with other cytokines or growth factors Include but are notlimited to: IFN γ or α, IFN-β; interleukin (IL) 1, IL-2, IL-4, IL-6,IL-7, IL-12, tumor necrosis factor (TNF) α, TNF-β, granulocyte colonystimulating factor (G-CSF), granulocyte/macrophage CSF (GM-CSF);accessory molecules, including members of the integrin superfamily andmembers of the Ig superfamily such as, but not limited to, LFA-1, LFA-3,CD22, and B7-1, B7-2, and ICAM-1 T cell costimulatory molecules.

The chemopreventive agent may precede or follow a DNA damaging agenttreatment by intervals ranging from minutes to weeks, Protocols andmethods are known to those skilled in the art. DNA damaging agents orfactors are known to those skilled In the art and means any chemicalcompound or treatment method that induces DNA damage when applied to acell. Such agents and factors include radiation and waves that induceDNA damage such as, gamma-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic agents”, function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-18), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide.

In another embodiment one may irradiate the localized tumor site withDNA damaging radiation such as X-rays, UV-light, gamma-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with the DNAdamaging agent by administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a DNAdamaging compound such as, adriamycin, 5-fluorouracil, etoposide,camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as gamma-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes.

As can be readily appreciated by one of ordinary skill in the art, themethods and pharmaceutical compositions of the present invention areparticularly suited to administration to a mammal, preferable a humansubject.

Intermediate endpoint biomarkers are measurable biologic alterations intissue that occur between the initiation of and the development of frankneoplasia. It is hypothesized that modulation of one or moreintermediate endpoint biomarkers by a chemopreventive agent may reflecttrue inhibition of carcinogenesis. A biomarker would be validated if thefinal endpoint, cancer incidence, were also reduced by the putativechemopreventive agent. Intermediate biomarkers in cancer may beclassified into the following groups: histologic, proliferation,differentiation and biochemical markers. In any chemopreventionstrategy, the availability of histologically recognizable and acceptedprecancerous lesions constitutes an important starting point. For theprostate, a possible histological marker is prostatic intraepithellalneoplasia (PIN), which is a precancerous precursor of prostaticadenocarcinoma. PIN appears as an abnormal proliferation within theprostatic ducts of premalignant foci of cellular dysplasia and carcinomain situ without stromal invasion. PIN and histological prostate cancerare morphometrically and phenotypically similar. Thus, the developmentof high grade PIN represents an important step in the progressionpathway whereby the normal prostate develops PIN, histological prostatecancer, invasive clinical prostate cancer, and metastases.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXPERIMENTAL DETAILS SECTION EXAMPLE 1 Transgenic Adenocarcinoma MouseProstate

The study of prostate cancer chemoprevention has been hindered by thelack of appropriate animal models. The recent development of thetransgenic adenocarcinoma mouse prostate (TRAMP) model enables the studyof chemoprevention. In the TRAMP model, which is described in Greenberget al., “Prostate cancer in a transgenic mouse,” Proc. Natl Acad. Sci.USA, 1995, Vol. 92, pages 3439-3443, the PB-SV40 large T antigen(PB-Tag) transgene is expressed specifically in the epithelial cells ofthe murine prostate. As a result, this model has several advantages overcurrently existing models: 1) mice develop progressive forms ofprostatic epithelial hyperplasia as early as 10 weeks and invasiveadenocarcinoma around 18 weeks of age; 2) the metastatic spread ofprostate cancer pattern mimics human prostate cancer with the commonsites of metastases being lymph node, lung, kidney, adrenal gland, andbone; 3) the development as well as the progression of prostate cancercan be followed within a relatively short period of 10-30 weeks; 4) thetumors arise with 100% frequency; and 5) the animals may be screened forthe presence of the prostate cancer transgene prior to the onset ofclinical prostate cancer to directly test treatment with chemopreventiveagents that may alter prostate carcinogenesis.

The TRAMP transgenic mouse model is an excellent in vivo model todetermine the mechanisms of initiation and promotion of prostate cancerand to test the effectiveness of potential chemopreventive agents. Thesemice progressively develop prostatic epithelial hyperplasia, PIN, andthen prostate cancer within a short period (<17 weeks).

Chemopreventive treatment of hybrid TRAMP mice is initiated 30 dayspostnatally, using chemopreventive agents at a level of about 0.5-50mg/kg of subject weight/day, preferably about 6-30 mg/kg of subjectweight/day. The chemopreventive agents are conveniently processed Into21-day and 90-day pellets (prepared by Innovative Research of America,Sarasota, Fla.) and delivered as subcutaneous implants. Control animalsreceive placebo implants. In each drug treatment group, animals aresacrificed at 5,7, 10, 15,20,25,30,40, and 50 weeks of age until thedevelopment of a palpable tumor. Blood is collected and pooled pertreatment time point to evaluate changes in serum testosterone andestradiol. Prostatic tissues are harvested for morphometric, histologic,and molecular studies.

The following test procedures are employed:

1) Prostate wholemount analysis is serially performed to detect changesin prostate ductal morphology over time with and without treatment;examples are shown in FIG. 2. Tissue sections are evaluatedhistologically by H&E and Masson-trichome standard staining. Theemergence of PIN is assessed and graded (I-mild to III-severe).

2) Serum estradlol and total testosterone levels are measured (RIA) foreach age interval to assess any changes in these hormones as a result ofchemopreventive agents.

EXAMPLE 2 Immunohistochemistry Data Analysis

Microscopy images of each tissue section are evaluated by usingcomputer-assisted (Mac 9500-I 32 computer and monitor) imagequantitation (NIH-Image 1.6 PPC) using Kodak DCS 460 camera on NikonMicrophot-FX microscope and quantitated by using a color-assistedquantitative system image analysis (IPLab Spectrum 3.1, Scanalytics,Inc., Virgina) that discriminates color differences of stained tissuesections. Thresholds are set to identify various tissue components ofthe prostate. The area pixel densities corresponding to each of thesetissue components are calculated for each full screen of the colormonitor. A total of 5 screens per prostate section are averaged.Immunohistochemical images can be digitalized and quantitated to enablestatistical evaluation by determination of sample correlationcoefficients and probability (2-tailed).

EXAMPLE 3 Study of Chemopreventive Activity

A study was undertaken to test the efficacy of chemopreventive agents inTRAMP transgenic animals (PBTag X FVBwt)(provided by Dr. NormanGreenberg, Baylor College of Medicine, Texas). These mice showedpreliminary signs of cancer as early as 10 weeks. The TRAMP transgenicmale litters were screened for the Large T ag transgene, and thepositive males were used in the study. The antiestrogen toremifene,which was to be tested for its possible chemopreventive effects, wasincorporated in customized pellets (Innovative Research of America,Sarasota, Fla.), and chemopreventive treatment of mice was initiatedpostnatally at 30 days (average mouse weight 14 g). Four groups of 10-12animals each received subcutaneous implantations of 90 day-releasetoremifene-containing pellets. The diffusible drug dosage, adjusted forgrowth related changes in weight, was designed to deliver either a lowdose (6 mg/kg) or a high dose (30 mg/kg) of toremifene. Control animals(n=10) received placebo implants. The efficacy of the treatment wasmeasured by the absence of palpable tumor formation. The murine prostatetumors were harvested and evaluated by molecular and histologicaltechniques.

Using the TRAMP transgenic model or prostate cancer, in which everyanimal that inherits the prostate cancer gene develops prostate cancer,it was demonstrated that toremifene both increases the latency anddecreases the incidence of prostate cancer.

As shown in FIG. 1 the effects of low and high dose toremifene were botheffective. Tumor formation in the TRAMP mouse ventral prostate was notedat week 17 for the placebo group (n=10), at week 19 for the high dosetoremifene-treated group(n=12), and at week 28 for the low dosetoremfene-treated group (n=12). Thus, 5 treatment by toremifenesubstantially increased the latency period by up to 11 weeks for thedevelopment of cancer in the ventral prostate of TRAMP mice.

Since the toremifene-treated animals did not reach the 50% tumordevelopment point during the period of the study, the time in which 25%of the animals had tumors was compared among groups. Tumors werepalpable in 25% of 10 the animals by week 23 in the placebo group and by30-31 weeks in the high and low toremifene groups, a delay of 7-8 weeks.Both low toremifene and high toremifene vs placebo were significant bylog rank and Wilcoxon statistical analysis, as shown in Table 1 below.

TABLE 1 Statistical Analysis Log-Rank Wilcoxon P p Low toremifene vsplacebo 0.0003* 0.0004* High toremifene vs placebo 0.0017* 0.0071**significance P < 0.05

At week 33, a point when all of the control animals had developedtumors, 72% of the low dose and 60% of the high dose toremifene-treatedanimals were still tumor-free. Thus, toremifene treatment at both lowand high dosages resulted in a greatly decreased incidence of tumors inthe ventral prostate of TRAMP mice.

These results, obtained in accordance with the present invention, wouldnot have been predicted from those reported in the aforementioned paperof Lucia et al., which describes the administering at two dosage levelsof tamoxifen, a close structural analog of toremifene, to Lobund-Wistarrats having prostate carcinomas induced by treatment with a combinationof an initiator and a promoter. In the Lucia et al. reference, it isreported that only 22-26% of the animals receiving the lower dose andonly 32-50% of those receiving the higher dose of tamoxifen remainedfree of tumors in the anterior prostate. It should be noted that theanterior prostate of a rodent, unlike its ventral prostate, has nocorresponding segment in the prostate of a human subject.

In Lucia et al., it is further stated that the initiator-promotercombination employed in the described procedures, although effective ininducing cancer in the anterior prostate of the test animals, failed toinduce carcinomas in the ventral prostate. Therefore there is no basisto expect a chemopreventive effect on tumors in the ventral prostate byadministering tamoxifen to Lobund-Wistar rats or to humans.

As already discussed, administering toremifene produces a substantialchemopreventive effect against tumors in the ventral prostate of TRAMPmice. This result is encouraging for a similar beneficial effect onhuman subjects, whose prostate does include a segment corresponding tothe ventral prostate of rodents.

EXAMPLE 4 Histological Examination of Prostate Tissue

Tumors from the placebo and high toremifene—treated groups taken at thetime of palpation were evaluated histologically. FIG. 2A is an H&Esection of the ventral prostate of a 17-week-old normal adult mouse.FIG. 2B, a section of the ventral prostate of a placebo-treated16-week-old TRAMP mouse, shows that, unlike the normal prostatestructure depicted in FIG. 2A, the TRAMP mouse ventral prostate ischaracterized by sheets of undifferentiated, anaplastic cells with ahigh mitotic Index. In contrast, as shown in FIG. 2C, the prostate of atoremifene-treated 30-week-old TRAMP mouse retains much of the normalglandular architecture and ha tumors with a more differentiatedstructure, the mitotic index being much lower than that for theplacebo-treated animal. These results indicate that toremifene, even atlow dosage, is able to suppress prostate carcinogenesis in the TRAMPmodel.

EXAMPLE 5 Use of Chemopreventive Efficacy of Toremifene Against ProstateCancer in the TRAMP Mouse Model

This experiment confirms and demonstrates the chemopreventive efficacyof toremifene. This present study focuses on the histological andmolecular changes associated with development of prostate tumor incontrol animals and the mechanism of toremifene chemopreventive actionwith TRAMP animals which are bred, screened and treated withsustained-release drug pellets. At predetermined times, groups of 5animals were sacrificed and their prostates were removed for analysis.The prostate glands were evaluated for the presence of tumor byhistology, wholemount dissections, and large T antigenimmunohistochemistry. To date, the Placebo and the Toremifene treatmentshave been completed for the 7, 10, 15 and 20 week time-points and theresults are described below.

Results: Prostatic wholemounts for 7, 10, 15, and 20 weeks for thevarious groups have been completed. Wholemount analysis revealed thatplacebo treated mice developed prostate tumors by 15-20 weeks of agesimilar to the previous pilot study. Moreover, the Toremifene treatedanimals had a delay in the occurrence of prostate cancer up to 20 weeks(FIG. 3). By 20 weeks, there is a striking delay in tumor occurrence inthe Toremifene treated group up to 35 weeks FIG. 4). These data confirmthat even with a more sensitive assessment of tumorigenicity, Toremifeneexhibited chemopreventive activity. For histological evaluation, tissuesamples were fixed, processed and paraffin embedded. Sections (5pMthick) were cut and stained by routine H&E method. Toremifene inhibitedthe ductal development and tissue differentiation (compare the 17 weeksTRAMP mouse prostate tumor vs. wildtype (FIG. 4); b) Toremifene treatedprostate histology vs. Placebo at 15 weeks (FIG. 5) Qualitatively,immunohistochemistry of Placebo and Toremifene treated tissues showedpresence of T-antigen in the ventral prostate. Thus, the chemopreventiveactivity seen by Toremifene does not appear to be by suppression of theprobasin promoter in the TRAMP model.

Conclusions: The ability of Toremifene to prevent the occurrence ofprostate cancer in the TRAMP model has been confirmed utilizing moresensitive techniques to assess tumor formation. The mechanism ofToremifene=s chemopreventive effects does not appear to be through lossof the transgene for the Large T-antigen protein.

EXAMPLE 6 Toremifene Induces Regression of Established Human ProstateCancer Tumors in the Nude Mouse Model

Prostate cancer currently remains the most commonly diagnosed cancer inAmerican males. However, questions remain about the etiology andtreatment of this disease especially is advanced forms. Hormone therapyremains the standard method of treatment for recurrent and advancedprostate cancer despite the common development of hormone refractorydisease. Therefore, new approaches for the prevention and treatment ofprostate cancer are needed to accommodate the increasing number of mendiagnosed with this disease. The experiments and results belowdemonstrate that toremifene suppresses hormone sensitive LNCaP tumorgrowth in athymic nude mice.

Materials and Methods: One million LNCaP cells in Matrigel weresubcutaneously injected into each flank of athymic nude mice. A total 40mice were injected. After approximately 34 weeks, visible tumorsdeveloped. After recording the tumor size in two dimensions, the micewere divided into placebo and treatment groups based on equivalent tumorburden. A single pellet (placebo versus toremifene 35 mg) wassubcutaneously implanted between the scapulae of each mouse. Weeklymeasurements of the tumor size were recorded. Tumor volume wascalculated (tumor volume=0.5 (L+W)×L×W×0.5236, where L=tumor length andW=width). The tumor volume at the time of pellet implantation served asthe point of reference for future comparison of that tumor=s sizevariation. The weekly variations of each tumor volume were recorded aspercent differentiation from the original measurement at pelletimplantation.

Results: Two mice died soon after pellet implantation due to mortalwounds from other mice. One mouse treated with toremifene was excludedfrom the study due to excessive tumor hemorrhage and hematomadevelopment. All mice developed visible tumors unilaterally orbilaterally. Each tumor was followed independently for the duration ofthe study. Twenty-four tumors were treated with placebo and 28 tumorswere treated with toremifene. The results are shown in Table 2 and FIG.6.

TABLE 2 PLACEBO GROUP Week N = % Change In volume relative to day 0 oftreatment 3 11 9.44 4 8 115.27 5 8 271.71 6 8 600.88

TOREMIFENE Week N = % Change in volume relative to day 0 of treatment 311 −34.58 4 7 −61.01 5 7 −74.51 6 5 −61.72

The follow-up interval will be extended on the currently reportedpopulation and data on additional animals are presently being collected.

Conclusion: Toremifene inhibits and induces regression of establishedLNCaP tumors. Although the mechanism by which toremifene exerts thiseffect is unknown, the ability to produce these effects supports the useof Toremifene as a treatment for prostate cancer and to prevent therecurrence of prostate cancer in high risk patients with establishedprostate cancer micrometastases.

EXAMPLE 7 The Role of Antiestrogens: Tamoxifen Citrate and Raloxifene(SERMs) and Faslodex (pure antiestrogen ICI 182,780) in the Preventionof Prostate Cancer

Experimental design: Chemopreventive treatment of mice are initiatedpost-natal at 30 days. Three groups of 50 hybrid TRAMP male mice eachare treated with either Tamoxifen citrate, or Raloxifene (SERMs) orFaslodex (pure antiestrogen ICI 182,780). The drugs is obtained ascustomized sustained-release pellets (Innovative Research of America,Sarasota, Fla.) and delivered as subcutaneous implants (see preliminarydata). Control animals are receive placebo implant with nopharmacological activity. Animals (n=10) are sacrificed at periodicIntervals, 10, 15, 20, 25 and 30 weeks age and the efficacy of thetreatment leading to either absence of tumor formation or reduction intumor size, if present, are assessed by comparison with placebo controlanimals. Blood is collected to evaluate changes in serum androgens andestrogens with each treatment. Prostatic tissues is saved for: a)morphometric studies; b) for histologic studies the tissue will be fixedin 10% buffered formalln, processed and paraffin embedded; c) formolecular studies the tissues is frozen in liquid nitrogen and stored at−70° C. Necropsies and survival data is also recorded.

The results of the experiment reveal the relative chempreventiveefficacy of the various antiestrogens in the delay or prevention ofprostate cancer in the TRAMP model. The morphological studies indicatethe gross changes, if any, in the development of the prostate size andductal pattern as a result of each treatment. Paraffinized tissuesections are stained using standard H&E techniques for histologicalchanges such as PIN that will be assessed to monitor the appearance ofprecancerous lesions as a precuror of prostatic adencarcinoma. Serumestradiol and total testosterone levels are measured for each ageinterval to assess any changes in these hormones, and whether or notthey correlate to changes in PIN. The peptide growth factor levels ofTGF, TGF 1, TGF 3, and bFGF is quantitative in prostate samples taken ateach interval. Corresponding peptide growth factor receptors is alsoassessed for EGFR and TGF RI and RII.

EXAMPLE 8 Toremifene Causes Regression of HGPIN in a Phase IIa ProstateCancer Chemoprevention Human Clinical Trial

The chemopreventive effects of an antiestrogen, toremifene againstprostate cancer have been reproducibly demonstrated herein in awell-established animal model of spontaneous human prostate cancer. Thisrepresents the first compound to demonstrate chemopreventive activityagainst prostate cancer. Moreover, High grade PIN (HGPIN) has now beenestablished and time tested as a precursor lesion for human prostatecancer also known as latent prostate cancer.

Consequently, HGPIN could be used as an intermediate endpoint, orsurrogate endpoint for human prostate cancer. In fact, the NCl has nowrecommended that PIN should be used as an intermediate endpoint, orsurrogate endpoint for human prostate cancer. In fact, the NCl has nowrecommended that PIN should be used as an intermediate endpoint.

A Phase IIa open labeled non randomized single center study with 20human subject is being conducted. In this protocol, patients with biopsyproven PIN are treated with 60 mg of Toremifene daily for 4 months.After 4 months, patients are rebiopsied and PIN status reassessed.Toremifene reduces PIN which thus will directly translate to a decreasein the incidence and a prolongation of the latency of prostate cancer.Other variables that are being investigated include serum PSA, qualityof life issues, and prostatic expression of TGFβ1 expression (thepresumed mechanism of action).

Currently, 9 patients have completed the study. All patents had highgrade PIN. HGPIN was determined from 8 biopsies from biopsy session 1which was confirmed 3 weeks later with 8 repeat prostate biopsy coresfrom biopsy session 2. After 4 months of Toremifene treatment (60mg/day), the patients were subjected to prostate biopsy session of 8prostate core biopsies from the same areas previously biopsied (left andright apex, midbase, base, and transition zones). The pathologicalevaluation revealed complete resolution of PIN with atrophic changes inthe prostatic epithelum. The patient experienced no acute or chronictoxicites while taking Toremifene. The serum PSA, serum freetestosterone, serum total testosterone, and serum estradiol remained inthe normal ranges. Quality of life was unchanged including no affect onpotency and libido. Therefore, these results demonstrate a prostatechemopreventive role for the antiestrogen toremifene.

What is claimed is:
 1. A method of suppressing or inhibiting latentprostate cancer of a subject comprising: administering to the subject, apharmaceutical preparation comprising an antiestrogen which does notform DNA adducts; and a pharmaceutically acceptable salts, esters, orN-oxides, or mixtures thereof, thereby suppressing or inhibiting latentprostate cancer of the subject.
 2. A method of treating a subject withprostate cancer, comprising: administering to the subject, apharmaceutical preparation comprising an antiestrogen which does notform DNA adducts; and a pharmaceutically acceptable salts, esters, orN-oxides, or mixtures thereof, thereby treating the subject withprostate cancer.
 3. The method according to any of claim 1 or 2, whereinsaid subject has benign prostatic hyperplasia, or an abnormally highlevel of circulating prostate specific antibody (PSA).
 4. The method ofclaim 1, wherein the subject has a precancerous precursors of prostateadenocarcinoma.
 5. The method of claim 4, wherein the precancerousprecursors of prostate adenocarcinoma is prostate intraepithelialneoplasia (PIN).
 6. The method according to any of claims 1 or 2,wherein said pharmaceutical preparation further comprises apharmaceutically acceptable carrier.
 7. The method according to claim 6,wherein said carrier is selected from the group consisting of a gum, astarch, a sugar, a cellulosic material, or mixtures thereof.
 8. Themethod according to any of claims 1 or 2, wherein said antiestrogen isadministered subcutaneously, orally, intravenously, intraarterially,intramuscularly, or topically.
 9. The method according to claim 8,whereby said subcutaneous administration is by implanting in saidsubject a pellet containing said pharmaceutical preparation.
 10. Themethod according to claim 9, wherein said pellet provides for controlledrelease of said pharmaceutical preparation over a period of time. 11.The method according to claim 8, whereby said intravenous,intra-arterial, or intramuscular administeration is by intravenously,intraarterially, or intramuscularly injecting in said subject saidpharmaceutical preparation in a liquid form.
 12. The method according toclaim 8, whereby said oral administration is by orally administering tosaid subject in a liquid or solid preparation containing saidpharmaceutical preparation.
 13. The method according to claim 8, wherebysaid topical administration is by applying to skin surface of saidsubject said pharmaceutical preparation.
 14. The method according to anyof claims 1 or 2, wherein said pharmaceutical preparation is selectedfrom the group consisting of a pellet, a tablet, a capsule, a solution,a suspension, an emulsion, an elixir, a gel, a cream, and a suppository.15. The method according to claim 14, wherein said suppository is arectal suppository or a urethral suppository.
 16. The method accordingto claim 15, wherein said pharmaceutical preparation is a parenteralformulation.
 17. The method according to claim 16, wherein saidparenteral formulation comprises a liposome comprising a complex of saidantiestrogen and a cyclodextrin compound.
 18. A method of suppressing orinhibiting pre-malignant lesions of prostate cancer of a subjectcomprising the steps of administering to the subject a pharmaceuticalcomposition comprising an antiestrogen which does not form DNA adducts;and a pharmaceutically acceptable salts, esters, or N-oxides, ormixtures thereof, thereby suppressing or inhibiting the pre-malignantlesions of prostate cancer of the subject.
 19. A method of treating asubject with pre-malignant lesions of prostate cancer comprising thesteps of: administering to the subject, a pharmaceutical compositioncomprising an antiestrogen which does not form DNA adducts; and apharmaceutically acceptable salts, esters, or N-oxides, or mixturesthereof, thereby treating the subject with pre-malignant lesions ofprostate cancer.
 20. The method of any of claims 18 or 19, wherein thepre-malignant lesion is a precancerous precursors of prostateadenocarcinoma.
 21. The method of claim 20, wherein the precancerousprecursors of prostate adenocarcinoma is prostate intraepithelialneoplasia (PIN).
 22. The method of claim 21, wherein the prostateintraepithelial neoplasia is high prostate intraepithelial neoplasia(HPIN).
 23. The method according to any of claims 18 or 19, wherein saidpharmaceutical composition further comprises an acceptable carrier ordiluent.
 24. The method according to claim 23, wherein said carrier isselected from the group consisting of a gum, a starch, a sugar, acellulosic material, or mixtures thereof.
 25. The method according toany of claims 18, 19, wherein said antiestrogen is administeredsubcutaneously, orally, intravenously, intraarterially, intramuscularly,or topically.
 26. The method according to claim 25, whereby saidsubcutaneous administration is by implanting in said subject a pelletcontaining said pharmaceutical composition.
 27. The method according toclaim 26, wherein said pellet provides for controlled release of saidpharmaceutical preparation over a period of time.
 28. The methodaccording to claim 25, whereby said intravenous, intra-arterial, orintramuscular administeration is by intravenously, intraarterially, orintramuscularly injecting in said subject said pharmaceuticalcomposition in a liquid form.
 29. The method according to claim 25,whereby said oral administration is by orally administering to saidsubject in a liquid or solid preparation containing said pharmaceuticalcomposition.
 30. The method according to claim 25, whereby said topicaladministration is by applying to skin surface of said subject saidpharmaceutical composition.
 31. The method according to any of claims 18or 19, wherein said pharmaceutical composition is selected from thegroup consisting of a pellet, a tablet, a capsule, a solution, asuspension, an emulsion, an elixir, a gel, a cream, and a suppository.32. The method according to claim 31, wherein said suppository is arectal suppository or a urethral suppository.
 33. The method accordingto any of claims 18 or 19, wherein said pharmaceutical composition is aparenteral formulation.
 34. The method according to claim 33, whereinsaid parenteral formulation comprises a liposome comprising a complex ofsaid antiestrogen and a cyclodextrin compound.